Color spectrophotometry uses a light source to illuminate a sample with light across the UV to the visible wavelength range (typically 190 to 900 nm). The instruments then measure the light absorbed, transmitted, or reflected by the sample at each wavelength. Some color spectrophotometry has an extended wavelength range, into the near-infrared (NIR) (800 to 3200 nm).
From the spectrum obtained, such as the one shown in the picture, it is possible to determine the chemical or physical properties of the sample. In general, it is possible to:
- Identify molecules in a solid or liquid sample.
- Determine the concentration of a particular molecule in solution.
- Characterize the absorbance or transmittance through a liquid or solid.
- Over a range of wavelengths.
- Characterize the reflectance properties of a surface or measuring the color of a material.
- Study chemical reactions or biological processes.
The key components of a color spectrophotometer are:
- A light source that generates a broadband of electromagnetic radiation across the spectrum.
- A dispersion device separates the broadband radiation into wavelengths.
- A sample area, where the light passes through or reflects off a sample.
- One or more detectors to measure the intensity of the reflected or transmitted radiation.
- Other optical components, such as lenses, mirrors, or fiber-optics, relay light through the instrument.
The ideal light source would yield a constant intensity over all wavelengths with low noise and long-term stability of the output. Unfortunately, such a source does not exist. Two different light sources have historically been used in color spectrophotometry:
The deuterium arc lamp was used to provide a good intensity continuum in the UV region and useful intensity in the visible region. The tungsten-halogen lamp yielded good intensity over the entire visible range and part of the spectrum.
An easy way to think about color spectrophotometry is to think of a room, with the sun shining through a window. The sunlight hits a prism that separates the white light into a rainbow. The rainbow falls onto a window on the opposite side of the room. As the prism is turned, the light of different colors, i.e. different wavelengths, passes out of the room through the window. Ideally, the output from color spectrophotometry is light of a single wavelength. In practice, however, the output is always a band of wavelengths. Most color spectrophotometry on the market today contain holographic gratings as the dispersion device. These optical components are made from glass, onto which extremely narrow grooves are precisely etched onto the surface. The dimensions of the grooves are of the same order as the wavelength of light to be dispersed. Finally, an aluminum coating is applied to create a reflective surface. Interference and diffraction of the light falling on the grating are reflected at different angles, depending on the wavelength. Holographic gratings yield a linear angular dispersion with wavelength and are temperature insensitive. However, they reflect light in different orders, which overlap (see the picture). As a result, filters must be used to ensure that only the light from the desired reflection order reaches the detector. A concave grating disperses and focuses light simultaneously.